The present invention relates to immobilized blue-green algae having enhanced growth and nitrogen-fixation rates. More particularly, the present invention relates to a multilayered structure incorporating such immobilized blue-green algae therein.
In recent years, there has been an increasing interest in the use of biological nitrogen fixation as a replacement for chemical nitrogen fertilizers. However, biological nitrogen fixation can be carried out only by a limited number of microorganisms. Foremost, perhaps, among such microorganisms are the blue-green algae, although not all blue-green algae are capable of fixing nitrogen. Such algae, i.e., the nitrogen-fixing blue-green algae, are able to fix nitrogen in an aerobic environment. Moreover, they are photosynthetic. For examples of references dealing generally with nitrogen fixation by blue-green algae, see H. W. Paerl, Can. J. Bot., 60, 2542 (1982); J. L. Ramos and M. G. Guerrero, Arch. Microbiol., 136, 81 (1983); J. S. Chapman and J. C. Meeks, J. Bacteriol., 156, 122 (1983); O. Ito and I. Watanabe, New Phytol., 95, 647 (1983); W. A. Wurtsbaugh and A. J. Horne, Can. J. Fish. Aquat. Sci., 40, 1419 (1983); P. M. Mullineaux et al., J. Gen. Microbiol. 129, 1689 (1983); Y. Chen, Zhiwu Shenglixue Tongxun 1983, 22; P. S. Tang et al., in C. K. Tseng, Editor, "Proceedings of the Joint China-U.S. Phycology Symposium," Science Press, Beijing, China, 1983, pp. 339-63; L. Leonardson, Oecologia. 63, 398 (1984); R. G. Elder and M. Parker, J. Phycol., 20, 296 (1984); L. J. Stal et al., Marine Biology, 82, 217 (1984); B. Bergman et al., Z. Pflanzenphysiol., 113, 451 (1984); and D. H. Turpin et al., Plant Physiol., 74, 701 (1984).
Under conditions of nitrogen deficiency, some of the vegetative cells of the algae differentiate into heterocysts which are capable of fixing atmospheric nitrogen. See, by way of illustration only, A. Kumar et al., J. Bacteriol., 155, 493 (1983); M. Roussard-Jacquemin, Can. J. Microbiol., 29, 1564 (1983); and references cited therein.
The use of blue-green algae as a biological nitrogen fertilizer is, of course, known. Such algae have been studied for or used in the cultivation of rice; see, e.g., G. S. Vankataraman and S. Neelakantan, J. Gen. Appl. Microbiol., 13, 53 (1967); W. D. P. Stewart et al., in "Nitrogen and Rice," International Rice Research Institute, Los Banos, Laguna, Philippines, 1979, pp. 263-85; G. S. Venkataraman in "Nitrogen and Rice," International Rice Research Institute, Los Banos, Laguna, Philippines, 1979, pp. 311-21; A. Agarwal, Nature., 279, 181 (1979); O. Ito and I. Watanabe, Soil Sci. Plant Nutr., 27, 169 (1981); G. S. Venkataraman, Current Science., 50, 253 (1981); G. S. Venkataraman, Trans. Int. Congr. Soil Sci. 12th, 2, 69 (1982); Z. T. Begum, Bangladesh J. Bot., 12, 127 (1983); L. Shanghao (S. H. Ley) and W. Qianlin, in C. K. Tseng, Editor, "Proceedings of the Joint China-U.S. Phycology Symposium," Science Press, Beijinq, China, 1983, pp. 479-96; A. Islam et al., Indian J. Agric. Sci., 54, 1056 (1984); V. Rajaramamohan Rao and J. L. N. Rao, Plant and Soil , 81, 111 (1984); B. S. Kundu and A. C. Gaur, Plant and Soil., 81, 227 (1984); I. Watanabe, Outlook on Agriculture, 13, pages unknown (1984); and H. C. Bold and M. J. Wynne, "Introduction to the Algae," Second Edition, Prentice-Hall, Inc., Englewood Cliffs, N.J., 1985, p. 37.
In addition, at least one company is marketing a blue-green algal fertilizer for the lawn and garden market. The fertilizer is prepared by blending dried algae with a soil-like carrier which allegedly prevents the dormant algae from dying. See B. R. Schlender, "New Uses for Algae Improve Image of a Lowly Plant Group," The Wall Street Journal, Friday, July 11, 1986, p. 25; Biotechnology News, Oct. 9, 1985, p. 6; and E. Eckholm, "Science Tries to Harness Bacterial Overachievers," The New York Times, Tuesday, Feb. 24, 1987, p. 15. Moreover, the application of blue-green algae to tomato plants reportedly resulted in 45% more growth (weight gain) than plants treated with the same amount of commercial fertilizer. It was postulated that the weight gain may be due to the secretion of a plant-growth hormone (Science/Technology Concentrates, Chemical and Engineering News, date unknown). Interestingly, cultivated soils apparently contain inconsequential numbers of blue-green algae; see W. Zimmerman et al., Soil Science, 130, 11 (1980).
In contrast with the large number of studies on the fixation of nitrogen by blue-green algae and the use of blue-green algae as a nitrogen fertilizer, little work apparently has been done with immobilized blue-green algae. Moreover, what work has been carried out was done primarily with entrapped cells, as described in the paragraphs which follow.
According to A. Muallem, Biotech 83: Proc. Int. Conf. Commer. Appl. Implic. Biotechnol. 1st 1983, pp. 1037-50, seven species of cyanobacteria, or blue-green algae, were entrapped in polyurethane foams. The entrapped cells, however, were used for the long-term photoproduction of hydrogen and NADPH.sub.2 from ascorbate and water. To immobilize the algal cells, pieces of foam were added to the culture vessels before autoclaving and inoculating with the algae. One species did not remain entrapped in any foam. In at least some cases, freeze-thaw cycles were employed as part of the immobilization procedure. The reference includes one electron micrograph, regarding which it was reported that short filaments and single cells were seen adhering to the polyurethane fibers which constitute the pore walls, and that cell envelope components were solely responsible for cell hydrophobicity which plays the major role in adhesion of benthic cyanobacteria on solid surfaces which have little or no surface charge. It is clear that no effort was made to measure nitrogen fixation by the immobilized algae. The immobilized cells apparently did not grow since they did not undergo any regenerative carbon metabolism.
Whole filaments of a nitrogen-fixing cyanobacterium were immobilized by entrapment in calcium alginate gel beads; S. C. Musgrave et al., Biotechnol. Lett., 4, 647 (1982) and Adv. Ferment. Proc. Conf. 1983, pp. 184-90. The immobilized cyanobacterium were used in various continuous-flow reactors for the sustained production of ammonia.
Finally, protein turnover in immobilized cells of a cyanobacterium was studied by M. Potts, J. Bacteriol., 164, 1025 (1985). The cells were immobilized by transfering a sample of a cell suspension to Whatman 3MM filter discs (23-mm diameter) which were supported on steel pins. The study employed only 50-microliter samples of a well-dispersed cell suspension and the algal filaments reportedly were immobilized immediately within the confines of the upper matrix of the support, occupying a circular area approximately 8 mm in diameter. The immobilization appears to be primarily an entrapment, and nitrogen fixation by the cells was not studied.
While blue-green algae were not involved, it perhaps should be mentioned that the continuous production of ammonia by an immobilized nitrogen-fixing-system depressed mutant strain of a bacterium, Klebsiella pneumoniae, has been reported; K. Venkatasubramanian and Y. Toda, Biotechnology and Bioengineering Symp. No. 10, 237-45 (1980). Immobilization involved mixing the cells with a collagen dispersion, adjusting the pH, casting a membrane, and crosslinking it with a mild solution of glutaraldehyde. Thus, the cells were entrapped in a collagen membrane.